Method for the purification of cytokeratin 20 and its use for the production of antibodies

ABSTRACT

In a process for the purification of cytokeratin 20 (CK 20), a cytoskeletal fraction of cells containing CK 20 is produced, the proteins present therein are separated by gel electrophoresis or/and by chromatography and the CK 20 is isolated from the gel or the chromatographic fraction containing CK 20. In a process according to the present invention for the production of antibodies specific for CK 20, purified CK 20 is used for the immunization and then polyclonal or monoclonal antibodies are produced according to well-known methods. These antibodies are used for the immunologically identification of CK 20, or its α-helical central fragment obtained by proteolytic cleavage on tissue sections, in tissue homogenates and in body fluids.

The invention concerns a process for the purification of cytokeratin 20,a standard protein material as well as a process for its production anda process for the production of antibodies directed against CK 20 andthe use of an antibody which is specific for CK 20 to detect thisprotein on tissue sections, in tissue homogenates or in body fluids, aswell as the use of a standard protein material to detect autoantibodiesagainst CK 20 in blood or serum.

It has been found that the intermediary filament (IF) proteins of thecytokeratin family are effective markers for analyzing the type andstate of differentiation of epithelial cells. The epithelialcytokeratins, comprising a family of at least 19 different polypeptides,are expressed in different combinations depending on the course of thecell differentiation. The synthesis of cytokeratins is usuallymaintained during malignant transformation and this fact could serve asone of the test criteria for epithelial-derived tumours, includingtumours of the bladder tract. There was inter alia a need for a reliabledetection method which can be easily carried out for determining thelocation of the primary tumour of metastastic tissue so that effectiveaction can be taken against this primary tumour. The object of thepresent invention was therefore to provide a way of differentiatingvarious types of tumour cells and detecting the origin of variousmetastastic tissues which can be carried out easily and as accurately aspossible.

A new cytokeratin has now been identified which only occurs inparticular cells and thus can serve as a marker to differentiate certaincells and tissues. It has been named cytokeratin 20.

The invention therefore concerns a process for the purification ofcytokeratin 20 in which a cytoskeletal fraction from tissues or cellscontaining CK 20 is prepared, the proteins present therein are separatedby gel electrophoresis or/and by chromatography and CK 20 is isolatedfrom the gel or from the chromatography fraction containing the CK 20.

The protein CK 20 has a molecular weight of ca. 46000, an isoelectricpoint in 9.5 molar urea of ca. 6.1 and is slightly more acidic thannon-phosphorylated variants of CK 8. FIG. 1 shows a partial amino acidsequence of CK 20 (denoted IT in this figure; A-D obtained fromfragments of CK 20 after digestion with Staphylococcus V8 protease).

According to the present invention it is possible to obtain cytokeratin20 in such a pure form that it is for example possible to use it toproduce specific antibodies. In one embodiment of the present inventionthe cytoskeletal fraction is produced from duodenal mucosal villi,especially from human tissue. In another embodiment of the invention thecytoskeletal fraction is isolated from culture cells wherein culturecells are preferred which are derived from colon carcinomas, bladdercarcinomas or stomach carcinomas. When carrying out a gelelectrophoresis in a preferred embodiment of the present invention afirst SDS polyacrylamide gel electrophoresis is carried out in a buffersystem with an increased salt concentration, subsequently the bandcontaining CK 20 is cut out and the protein is eluted from it and asecond polyacrylamide gel electrophoresis is carried out in a buffersystem with a lower salt concentration and the now purified CK 20 isisolated from the corresponding band in the gel.

In a further embodiment of the invention, namely separation bychromatography, an anion-exchange chromatography is firstly carried outand then a reversed phase HPLC chromatography. In this case it is inturn preferred that the anion-exchange chromatography is carried out onDEAE cellulose in the presence of urea and using an eluant with a lineargradient of between 0 and 100 mmol/l guanidinium hydrochloride and tosubsequently subject the fractions containing CK 20 to HPLC. In thisprocess the fractions containing 38 to 50 mmol/l guanidiniumhydrochloride are preferably subjected to HPLC.

The invention furthermore concerns a standard protein material whichconsists of a reconstituted cytokeratin complex containing CK 20 and abasic cytokeratin from the group of cytokeratins 1 to 8 or consists ofthe corresponding α-helical central fragments of these proteins producedby proteolytic cleavage. In this connection it is preferred that thestandard protein material consists of a complex containing CK 20 and CK8 or their α-helical central parts.

Such a standard protein material is a suitable agent for use inimmunological tests, for example when employing the displacementtechnique or for carrying out other competitive immunoassays.

The invention in addition concerns a process for the production of astandard protein material containing CK 20 and a basic cytokeratin inwhich purified CK 20 and a purified basic cytokeratin from the group ofthe cytokeratins 1 to 8 are together dissolved in an equimolar ratio ina buffer containing urea and the mixture is firstly dialysed against abuffer containing urea and DTT and then against a buffer without urea. Areconstituted filament material is obtained in this process which formswhen urea is removed from the buffer. In a preferred embodiment of theinvention the cytokeratin complex which is formed is cleavedproteolytically after the production of the standard protein material.The α-helical central fragments of the reconstituted standard proteinmaterial are obtained in this manner. This proteolytic cleavage ispreferably carried out with chymotrypsin and in an enzyme to substrateratio of 6:1000 to 10:1000 and digestion times between 30 and 60minutes.

It is particularly preferred according to the present invention that CK8 be used as the basic cytokeratin in the production of the standardprotein material. It is also in turn preferred that a purified CK 20 beused which has been purified according to the process according to thepresent invention. In order to produce a standard protein material inanother preferred embodiment of the invention, the proteins aredissolved in a buffer containing 8.5 to 10 mol/l urea and 1.5 to 3mmol/l DTT and the first dialysis is carried out against a buffercontaining 3.5 to 4.5 mol/l urea and 1.5 to 3 mmol/l DTT.

The invention in addition concerns a process for the production ofantibodies specific for CK 20 by using purified CK 20 to immunizesuitable animals and then produce polyclonal or monoclonal antibodiesaccording to known methods. The basic production of polyclonalantibodies as well as of monoclonal antibodies is known to a personskilled in the art and the production of monoclonal antibodies is forexample described by Kohler and Milstein in Nature 256 (1975) 495-497.Guinea-pigs are preferably immunized with the purified CK in order toobtain polyclonal antibodies.

In a preferred embodiment of the present invention, CK 20 purifiedaccording to the present invention is used.

In order to actually obtain monospecific antibodies which are directedagainst CK 20 during the production of polyclonal antibodies, it is inturn preferred according to the present invention that thesemonospecific antibodies are isolated by subjecting the immunoglobulinfraction to an immunoprecipitation and separating antibodies directedagainst other cytokeratins or/and to an immunoprecipitation andisolating the antibody specific for CK 20. For this purpose allimmunoprecipitations known to a person skilled in the art are suitableand finally an antibody is obtained which only reacts immunologically toCK 20. In a preferred embodiment of the present invention, theimmunoprecipitation and isolation of antibodies specific for CK 20 iscarried out by incubating the immunoglobulin fraction obtained from theexperimental animal with a solid phase to which CK 20 has been coupled.When antibodies directed against other cytokeratins areimmunoprecipitated and separated, it is preferable to incubate theimmunoglobulin fraction obtained with a solid phase to which theelectrophoretically purified cytokeratins 8, 18 and 19 or total proteinobtained from these cells has been coupled. According to the presentinvention the immunoprecipitation steps are preferably carried outseveral times so that all antibodies which are not directed against CK20 are actually separated from the monospecific antibodies. In turn itis preferred according to the present invention that nitrocellulosestrips be used as the solid phase.

The invention furthermore concerns the use of an antibody which isdirected against CK 20 for the immunological identification of CK 20 orits α-helical central fragment obtained by proteolytic cleavage ontissue sections, in tissue homogenates and in body fluids. In thisconnection it is in turn preferred that a homogenate is made from atissue sample, the intermediary filament proteins present in thehomogenate are proteolytically cleaved and the α-helical centralfragments released from this into the soluble phase are isolated andquantitatively determined and identified with the aid of the antibody.

A further preferred embodiment of the present invention allows thesoluble intermediary filament protein fragments present in body fluidssuch as blood, blood serum and urine to be immunologically identifiedand quantitatively determined using the antibody.

The use of the antibody against CK 20 according to the present inventionallows a determination of whether this protein is present in tissues,tissue homogenates or body fluids. The presence of this protein enablescells or tissues to be differentiated with respect to the occurrence ofCK 20 in particular tissues. Thus adenocarcinomas of thegastrointestinal tract, of the bladder and of Merkel cells of the skincan for example be distinguished from other adenocarcinomas and it isalso possible to determine the cellular origin of metastases; hence theCK 20 protein test enables metastases which may be found at completelyunrelated sites in the body to be assigned to a primary tumour of one ofthe aforementioned regions. This allows the actual source of the tumourto be acted on therapeutically by identifying the primary tumour whichis often extremely difficult or is not possible to locate at all bypreviously known methods and thus allows the chances of survival for thepatients to be considerably increased. Such a method for distinguishingadenocarcinomas of the gastrointestinal tract, of the bladder or ofMerkel cells from other adenocarcinomas or the test for the cellularorigin of metastases by examining the presence of CK 20 in the tissue tobe examined using antibodies according to the invention which arespecific for CK 20 is therefore a further subject matter of the presentinvention.

Yet another subject matter is the use of a standard protein materialaccording to the present invention to detect autoantibodies against CK20 in blood or serum. Autoantibodies against CK 20 are for exampleformed during chemotherapeutic treatment of tumours and can be rated asa criterium for the progress of treatment. Furthermore, suchautoantibodies can also be formed in other diseases, e.g. in MorbusCrohn, by which means it is also possible to use the standard proteinmaterial to substantiate or to disprove an indication for a disease.

In addition the standard protein material can, as already mentioned, beused in immunological tests which use the antibody according to thepresent invention to detect CK 20 when for example carrying out animmunoassay of the displacement type.

Immunoassays which can be carried out with the antibodies or thestandard protein material according to the present invention are knownto a person skilled in the art and do not need to be described in moredetail here. In this connection it is of course obvious that thedetection is carried out by means of any type of labelling whatsoever ofantibodies or standard protein material whereby in this case it appearsto be possible to use all known actual test procedures.

The antibody according to the present invention can also be used tocarry out a test for cell lesions when this is in an appropriate format,such as that described for example in EP-A 0 057 043. The antibodyaccording to the present invention can also be used to carry out aprocess like that described in EP-A 0 057 076 in an analogous manner forcytokeratin 20. In this case the antibody according to the presentinvention is used as the antibody which definitely reactsimmunologically.

It is intended to further elucidate the invention by the followingexamples in conjunction with the figures.

BRIEF DESCRIPTION OF THE DRAWING

In this connection FIG. 1 shows the partial amino acid sequence of CK 20fragments which were obtained by digestion with Staphylococcus V8protease and fractionation by reverse phase HPLC. These sequences arecompared to the corresponding sequences of various human type Icytokeratins. Amino acids which are identical are shown in bold print.Dots denote omissions which were made in order to show correlationsbetter.

EXAMPLE 1 Production of a Cytoskeletal Preparation

A cytoskeletal fraction was prepared on a large scale from duodenalmucosal villi. The deep-frozen villous material was thawed, homogenizedby means of a Polytron homogenizer (Kinematica, Luzern, Switzerland) andextracted for 20 minutes while stirring on ice with a five-fold volumeof buffer A (1.5 mol/l KCl, 0.5% Triton X-100, 5 mmol/l EDTA, 0.4 mmol/lphenylmethylsulfonyl fluoride (PMSF), 10 mmol/l Tris-HCl, pH 7.2. Acytoskeletal pellet was obtained by centrifugation (10 minutes at 13000g, 4° C.) and washing the sediment twice (resuspending by means of aDounce homogenizer and centrifuging again) in buffer B (5 mmol/l EDTA,0.4 mmol/l PMSF, 10 mmol/l Tris-HCl, pH 7.2); this was stored at -80° C.Cytoskeletal fractions were also prepared from other tissues and tumoursin an analogous manner; in these cases the tissue was cut up into smallpieces using scissors or a scalpel before the Polytron homogenization.

Cytoskeletal fractions from culture cells were obtained by a similarprocedure. Cells which grew adherently at the bottom of plastic cultureflasks were scraped off using a rubber spatula after decanting theculture medium and rinsing twice with phosphate-buffered saline solution(PBS), extracted in buffer A and subsequently washed with buffer B (seeAchtstatter et al., Methods Enzymol. 134 (1986) 355-371).

Culture Cells

The following established cell culture lines derived from humancarcinomas were used in the investigations:

1. The bladder carcinoma cell lines RT-112, RT-4, T-24 and EJ (see Mollet al., Am. J. Pathol. 132 (1988) 123-144).

2. Several colonic carcinoma cell lines obtained from the American TypeCulture Collection (ATCC) and cultured as stated there: HT-29 (ATCC HTB38); LoVo (ATCC CCL 229); SW 1116 (ATCC CCL 233); LDL-1 (ATCC CCL 221);COLO 320 DM (ATCC CCL 220).

3. Cells of the human mammacarcinoma cell line MCF-7 were cultured asdescribed by Moll and coworkers (Cell 31 (1982) 11-24). In someexperiments culture cells were labelled metabolically with ³⁵S-methionine (Franke and coworkers, Knapp and Franke, Cell 59 (1989),67-79. "Spontaneous losses of control of cytokeratin gene expression intransformed, non-epithelial human cells occurring at different levels ofregulation").

EXAMPLE 2 Preparation of Pure CK 20

In order to obtain CK 20 proteins, two different methods for isolatingproteins were used. On the one hand, preparative gel electrophoresis wasused (Achtstatter and coworkers, 1986, see example 1) in whichcytoskeletal proteins obtained from duodenal mucosal villi werefractionated in a one-dimensional electrophoresis (SDS-PAGE) andvisualized by sodium acetate staining. The elution of the protein fromthe bands which were cut out was either carried out electrophoreticallyor by incubation of the finely homogenized gel material in a 0.05%aqueous SDS solution. The eluted protein was concentrated by means ofvacuum dialysis and precipitated with acetone. The Laemmli system(Laemmli, U.K., Nature 227 (1970) 680-685) or the buffer system with anincreased salt concentration (Achtstaetter and coworkers, 1986 (seeabove)) was used as the SDS-PAGE system and in some cases both were usedsuccessively.

A combination of DEAE-cellulose anion-exchange chromatography and"reverse phase" HPLC chromatography was used as a second method with thesame starting material (Achtstaetter and coworkers, 1986, see above).The purity of the protein fractions was tested by means of SDS-PAGE. Thechromatographic method requires no SDS denaturation and was thereforethe method of choice for in vitro experiments of complex formation andreconstitution.

EXAMPLE 3 Production of Specific Polyclonal Antibodies Against CK 20

CK 20 protein purified by preparative gel electrophoresis was used toimmunize mice and guinea-pigs using the immunization model of Franke andcoworkers (Franke, Denk, Kalt and Schmid (1981) Biochemical andimmunological identification of cytokeratin proteins in hepatocytes ofmammalian liver tissues. Exp. Cell Res. 131, 299-318). It was possibleto isolate an antiserum which contained high titres of antibodiesagainst CK 20 protein but also antibodies against CK 18. Monospecificantibodies against CK 20 protein could be isolated from this serum(without reactivity to CK 18) by absorbing the serum diluted in PBScontaining 1% bovine serum albumin and 0.1% sodium azide (or animmunoglobulin fraction produced from this by ammonium sulfateprecipitation) several times on nitrocellulose strips to whichelectro-transferred cytokeratins 8, 18 and 19 from MCF 7 cells or fromMCF 7 cell total protein which had been fractionated by SD-SPAGE werebound. The absorption was carried out in each case by incubating for 30minutes in a small foil sack which was rotated continuously. Between theabsorption steps the nitrocellulose strips were regenerated byincubation in 3 mol/l KSCN in PBS and subsequent washing in PBS. Aboutfour to eight absorption steps were carried out in succession. In someexperiments a positive affinity purification step was carried out onnitrocellulose strips with CK 20 protein separated by means of SDS-PAGEbefore this counter-absorption; for this the antibodies bound to thesestrips were eluted by means of 3 mol/l KSCN in PBS and vacuum dialyzedagainst PBS (Krohne and coworkers, J. Cell Biol. 94 (1982) 749-754). Thespecificity of the purified antibody preparations was assured byimmunoblot analysis after two-dimensional gel electrophoreticfractionation.

EXAMPLE 4 In Vitro Reconstitution of Heterotypical Cytokeratin Complexesand Intermediary Filaments

Chromatographically-purified proteins (CK 8, 18 and 20) obtained fromcytoskeletal material of duodenal mucosal villi were dissolved in a 10mmol/l Tris-HCl buffer (pH 8.0) which contained 2.5 mmol/l DTT and 9.5mol/l urea and (after centrifuging residual insoluble material at 13000g) either dialyzed singly or in certain approximately stoichiometricmixtures (CK 8+CK 18; CK 8+CK 20) against the same buffer containing DTTbut only 4 mol/l urea. By this means it should be possible to achieve aheterotypical complex formation between type I and type II cytokeratins.Aliquots of the solution adjusted to 4 mol/l urea (after centrifugation)were directly subjected as samples to an electrophoresis undernon-dissociating conditions in 4 mol/l urea combined with SDS-PAGE inthe second dimension.

For the in vitro reconstitution of intermediary (cytokeratin) filaments,chromatographically-purified CK 8 and CK 20 protein were each dissolvedat a concentration of 1 mg/ml (protein determination according toBradford (Bradford M.M., Anal. Biochem. 72, (1976), 248-254)) (buffersee above) and mixed in an equimolar ratio. This mixture (and alsosolutions of the individual proteins as a control) was dialysed for onehour on swimming membrane filters (Millipore VS 0.025) against 4 mol/lurea in 10 mmol/l Tris-HCl (pH 7.6) containing 2.5 mmol/l DTT andsubsequently dialysed for two hours against 10 mmol Tris-HCl (pH 7.6)containing 2.5 mmol/l DTT but no urea. Subsequently, negative stainingand electron microscopic examination were carried out (Quinlan et al.,J. Mol. Biol. 178 (1984), 365-388).

EXAMPLE 5 Proteolytic Cleavage Experiments

Native cytoskeletal material from duodenal mucosal villi was subjectedto a partial proteolytic (chymotryptic) cleavage (Hatzfeld andcoworkers, J. Mol. Biol. 197 (1987) 237-255). Enzyme-substrate ratios of6.6:1000 or 9:1000 and digestion times between 30 and 60 minutes wereused in this case. The cleavage products were analyzed by means ofSDS-PAGE with subsequent silver staining or immunoblot analysis or bymeans of two-dimensional gel electrophoresis combined with a trypticpeptide mapping.

EXAMPLE 6 Immunocytochemistry, Methods Used for the Immunofluorescence

Immunoperoxidase and immunoelectron microscopy of cryostatic tissuesections and culture cells were applied as described (Bader et al., Eur.J. Cell Biol. 47 (1988) 300-319; Franke et al., Proc. Natl. Acad. Sci.USA 75, (1978) 5034-5038; Franke et al., Exp. Cell Res. 116 (1978)429-445; Franke et al., Eur. J. Cell Biol. 19 (1979) 255-268; Franke etal., Exp. Cell Res. 131 (1981) 299-318; Franke et al., J. Cell Biol. 90(1981) 116-127; Franke et al., Cell 30 (1982) 103-113; Franke et al.,Virchows's Archiv A, Pathol. Anat. 411 (1987) 137-147; Jahn et al.,Differentiation 36 (1987) 234-254; Knapp and Franke, Cell 59, (1989)67-79, Moll et al., Am. J. Pathol. 132 (1988) 123-144).

EXAMPLE 7 Method for the Isolation of a Standard

The methods used are described using cytokeratins as an example; theprocessing technique can, however, be applied to all IF proteins.

7.1 Purification of intact polypeptides

Human cytokeratins (e.g. 8) were isolated from the human culture cellline MCF-7 essentially as described by Achtstaetter et al., MethodsEnzymol. 134:355-371 (1986). The cell layer which was scraped off ishomogenized (essentially as described by Achtstaetter et al., supra).Individual cytokeratin polypeptides were purified chromatographically bymeans of anion-exchange chromatography on DEAF-cellulose (DE 52; WhatmanChemical Separation Inc., Clifton, N.J., U.S.A.) in a 8 mol/l (forcytokeratin 8) or 9.5 mol/l (for cytokeratin 20) urea buffer (8 or 9.5mol/l urea, 2.5 mmol/l dithioerythritol, 30 mmol/l Tris-HCl (pH 8.0)),essentially as described by Hatzfeld & Franke, J. Cell. Biol. 101 (1985)1826-1841; Achtstaetter et al., 1986 (see above); Bader et al., EMBO J.5 (1986) 1865-1875; Quinlan et al., J. Mol. Biol. 192 (1986) 337-349.Briefly described: cytoskeletal material was extracted for 2 hours in9.5 mol/l urea (5 mmol/l dithioerythritol, 10 mmol/l Tris-HCl (pH 8.0))and the supernatant extract which was obtained after centrifugation at100,000×g (g=gravitational constant) was dialysed against a 8 or 9.5mol/l urea buffer and applied to a DEAE-cellulose column which had beenequilibrated with this buffer. Bound protein was eluted with a 0 to 100mmol/l guanidinium-HCl gradient. The polypeptide composition wasmonitored by SDS-polyacrylamide gel electrophoresis (PAGE). The combinedfractions were subjected to a further purification by a reverse-phasehigh pressure liquid chromatography using 0.01% (v/v) trifluoroaceticacid (TFA) (Fluka, Buchs, Switzerland) as the aqueous solvent A, 0.07%(v/v) TFA in acetonitrile (chromatographic quality, Merck Darmstadt,GFR) as the organic phase (solvent B) and a reverse phase BioRad RP 304column (BioRad Laboratories, Richmond, Calif., U.S.A.). The peakfractions were pooled, the acetonitrile was removed by evaporation in avacuum and the fractions were freeze-dried.

7.2 Reconstitution of purified polypeptides to form protofilaments andIF proteins

The purified cytokeratins were dissolved in buffer containing 9.5 mol/lurea. Equimolar amounts of type I and type II cytokeratin were mixed ata final concentration of about 0.5 mg/ml and the protofilaments andcytokeratin filaments were obtained by dialysing the polypeptidesolution against buffer of a low ionic strength. (The buffers describedby Hatzfeld, M. and Franke, J. Cell Biol. 101: 1826-1841 (1985) wereused). The formation of protofilaments and cytokeratin filaments wasmonitored by electron microscopy using negative sample contrast (cf.Hatzfeld and Franke, supra).

7.3 Preparation of α-helical central fragments of cytokeratin by limitedproteolysis

The proteolytic degradation was carried out with various proteases. In atypical preparation, chymotrypsin (EC 3.4.21.1 from bovine pancreas(e.g. from the Sigma Company, Munich) is used in an enzyme to substrateratio (weight/weight) of 6.6:1000 for the cytokeratin 8:18 pair and in aratio of 9:1000 for the cytokeratin 8:20 pair. The digestion time had tobe optimized for each chymotrypsin batch. The proteolytic degradationwas monitored by gel electrophoretic analysis of the degradationproducts and optimized for a maximum proportion of rod-shaped centralfragments (M_(r) =38000-40000). The optimum is ca. 20 min at 30° C.After the appropriate degradation period,the enzyme activity was stoppedby addition of 5 mM phenylmethylsulfonyl fluoride.

7.4 Purification of α-helical central fragments

The proteolytic central fragments and their single fragments were eitherseparated chromatographically on a Sepharose CL6B (Pharmacia LKB,Freiburg) column or directly by reverse-phase high performance liquidchromatography and namely on a reverse-phase BioRad RP304 column usingthe solvent system described in the above section 7.1. For furtherpurification, the main fractions were dissolved with solvent A in orderto reduce the acetonitrile concentration to ca. 20% (v/v) and thenapplied directly to a My-Bondapak reverse-phase C18 column (WatersAssociates, Milford, Mass.). All main fractions were lyophilized and thesamples were examined by means of one- and two-dimensional gelelectrophoresis for the presence of α-helical central fragments some ofwhich are divided once into two fragments of the central fragment sincethe cleavage site is located in a short central section where thehelical structure is interrupted, and they were used as referencematerials and standard materials for calibrating the detection systemand for immunizations.

EXAMPLE 8 Selection and Production of Suitable Antibodies

The following methods were used to examine specific antibodies againstintermediary filament proteins developed by us and correspondingcommercial antibodies for their immunoreactivity with the α-helicalfragments of the central part which were obtained as standard materialaccording to example 7:

8.1. Immunoblot (Western blot; reaction with denatured antigen)

Purified fragment proteins (e.g. cytokeratin 8:18 fragments, cytokeratin8:20 fragments or vimentin fragments) and cytoskeletal preparations fromtissue samples (e.g. lymph nodes or liver) were separated gelelectrophoretically (sodium dodecylsulfate polyacrylamide gelelectrophoresis) before and after a proteolytic digestive reaction withchymotrypsin (compare example 9), the proteins were transferredelectrophoretically onto nitrocellulose and incubated with possiblespecific antibodies. The immune reaction was detected via labelledprotein A or labelled anti-mouse antibodies. Antibodies which showed animmune reaction with the α-helical central fragments (M_(r) 38000-40000;M_(r) 20000-22000 in the case of basic keratins) were selected.

8.2 Dot-blot (reaction with native or renatured antigen)

Ca. 2×10⁻⁶ g purified CK 20 proteins (dissolved in 50×10⁻⁶ l 50 mmol/lNa₂ HPO₄ buffer, pH 7.4) and supernatant fractions of homogenized tissuesamples after digestion with chymotrypsin are directly bound tonitrocellulose (e.g. in a SRC 96 Minifold I dot-blot apparatus fromSchleicher and Schuell, Kassel GFR) and incubated with possible specificantibodies. The further procedure is as described under 1.

8.3 ELISA (reaction with native or renatured antigen)

500 ng (10⁻⁹ g) purified fragment proteins (dissolved in 100 μl (10⁻⁶ l)50 mmol/l NaHCO₃ buffer, pH 9.6) and 2 μg (10⁻⁶ g) (in 100 μl [10⁻⁶ l])protein from the supernatant fractions of homogenized tissue samplesafter digestion with chymotrypsin are incubated per well in a 96-wellmicrotitre plate and bound protein is incubated with the possiblespecific antibodies. The further procedure is as described under 1.

8.4 Immunofluorescence microscopy. Standard method, described in detaile.g. by Ciocca D. R. and Bjercke R. J. (1986) in Methods Enzymol. 121,562-579.

Antibodies and antisera which were positive according to theabove-mentioned methods are K_(s) 19.2; K_(s) 18-9B1; K_(s) 18-27 IV;K_(s) 8-17.2; K_(s) pan 1-8; VIM 3B4; IT guinea-pig antiserum(production of guinea-pig antiserum against cytokeratin 20, see example3), IT mouse antiserum (production see example 3).

8.5 Production of monoclonal antibodies directed against α-helicalcentral fragments

In order to produce specific antibodies only in vitro reconstitutedfilaments formed from the corresponding purified polypeptides wereinjected for the immunization. In order to produce monoclonalantibodies, female 6-8 week old BALB/c mice were immunized by injectingthem with cytoskeletal preparations or reconstituted filaments with30-300×10⁻⁶ g protein per injection. The antigens were suspended inphosphate-buffered saline solution (PBS) and emulsified with Freund'sadjuvant (complete) for the first injection. In all the followinginjections Freund's adjuvant (incomplete) was used. The animals wereinjected subcutaneously three times at intervals of about three weeksand they received an intraperitoneal booster injection of 30-80×10⁻⁶ gantigen three days before cell fusion. The spleen cells of immunizedmice were fused essentially as described by Koehler and Milstein, Nature256: 495-497 (1975) with mouse myeloma cells of the line Sp3/OAg14,X63-Ag8.653 and NSO/U (described by Shulmann et al., Nature 276: 269-270(1978); Kearney et al., J. Immunol. 123: 1548-1550 (1979); Clark andMilstein, Somatic Cell Genetics 7: 657-666 (1981)) in a ratio of 10:1.The hybridoma supernatants were tested on frozen sections of human andbovine tissue by means of immunofluorescence microscopy (essentially asdescribed by Achtstaetter et al., Differentiation 31:206-227 (1986)) oron culture cells which were cultured on specially coated glass slides orcoverglasses or tested using the enzyme-linked immunoadsorptiontechnique (ELISA) in which the purified antigens were used to coatmicrotitre plates. Positive clones were subcloned twice by means ofcontrolled dilution. Ig subclasses were determined according toOuchterlony and Nilsson, L. A. (1978 in: Handbook of ExperimentalImmunology; Wei Ed., Vol. 1, chapter 19, Blackwell ScientificPublications, Oxford, pp. 1-19).

8.6 Coupling of detector antibodies to peroxidase.

The monoclonal antibodies denoted detector antibodies and specificguinea-pig antiserum were coupled to peroxidase according to a methoddescribed by B. Tijssen (Laboratory techniques in biochemistry andmolecular biology, Vol. 15: Practice and theory of enzyme immunoassays,R. H. Burdon and P. H. van Knippenberg, eds., Elsevier Amsterdam, NewYork, Oxford; p. 238):

5 mg peroxidase is dissolved in 0.5 ml sodium carbonate buffer (100mmol/l, pH 9.2) and prepared for the coupling by oxidizing the enzymefor 2 hours at room temperature and in complete darkness with 0.5 ml ofa 10 mmol/l NaIO₄ solution. Afterwards the desired antibody (10 mgdissolved in 2 ml 100 mmol/l sodium carbonate buffer, pH 9.2) is added,0.5 g dry Sephadex G-25 (Pharmacia Co., Freiburg) is added and it isincubated for a further 3 hours in complete darkness. The conjugatewhich formed in this process is eluted from the Sephadex material usingthe sodium carbonate buffer and mixed with 1/20 parts by volume of a0.5% NaBH₄ solution in 0.1 mmol/l NaOH. 30 minutes later 1/10 parts byvolume of the same solution is added and incubated for 1 hour at 4° C.The conjugate is vacuum-dialysed against PBS and concentrated to ca. 0.5ml and subsequently fractionated on a Sephadex-G-200 column (PharmaciaCo.) (1.0×50 cm). The fractions (ca. 0.5 ml volume) are tested for theirproportion of enzyme activity and antibodies, and the fractions whichhave a concomitant high Ig concentration and high enzyme activity arepooled.

EXAMPLE 9 Detection and Determination of Metastases in Lymphatic Tissue

9.1 Solubilization of CK 20 proteins and preferably their α-helicalcentral fragments

Firstly the wet weight of the lymph node tissue is determined. Thetissue is ground in a three-fold volume--in relation to the wetweight--of a phosphate-buffered saline solution (PBS) (10 mmol/l sodiumphosphate pH 7.4, 150 mmol/l sodium chloride) using a knife homogenizeruntil a pulp-like consistency is achieved (the use of a Polytronhomogenizer from the Kinematica Company, Luzern/Switzerland isrecommended). The homogenate is incubated with chymotrypsin.

For this purpose chymotrypsin is used which has previously been bound toa matrix (CNBr-activated Sepharose 4B, Pharmacia Co., Freiburg): 1 gCNBr-activated Sepharose 4B is swollen for 15 min in 1 mmol/l HCl (gelvolume ca. 3.5 ml/g) and washed with a total of 200 ml 1 mmol/l HCl. Thehydrochloric acid solution is aspirated and the matrix material iswashed with 5 ml coupling buffer (0.5 mol/l NaCl, 0.1 mol/l NaHCO₃, pH8.0). 10 mg chymotrypsin is dissolved in 5 ml coupling buffer andincubated for 2 hours at room temperature with the matrix material incoupling buffer while agitating continuously. Unsaturated coupling siteswhich remain are subsequently blocked by addition of 5 ml 0.2 mol/lglycine solution (pH 8.0). Subsequently the gel material is washed withan excess of coupling buffer (ca. 50 ml) and 10 ml acetate buffer (0.5mol/l NaCl, 0.1 mol/l sodium acetate, pH 4.0). Ca. 60% of thechymotrypsin used is bound under these conditions, i.e. the Sepharose 4Bgel contains 1.7 mg/ml coupled chymotrypsin. The gel is diluted two-foldin PBS for better handling (chymotrypsin concentration 0.85 mg/ml).

Chymotrypsin (EC 3.4.21.1 from bovine pancreas, e.g. from the Sigma Co.Munich) is added to the tissue pulp in a ratio of 1:1000 (calculated forthe wet weight of the tissue). This is followed by an incubation step at30° C. (preferably in a thermal block, eventually in a water bath). Thedigestion reaction is stopped after 30 minutes by placing the homogenateon ice for 5 min. The homogenate is centrifuged for 30 min at 2×10⁴ gand the centrifugation supernatant is immediately removed; the coupledchymotrypsin is located in the sediment.

Under these conditions 80 to 95% by weight of the α-helical centralfragment material is released from the CK 20 proteins into thesupernatant fraction in a state which is still identifiable and someintact CK 20 proteins are also in a soluble state.

9.2 Detection and determination of the vimentin content

Vimentin in the centrifuged supernatant is determined immunologically bya sandwich ELISA. For this, a first antiserum, GP-8 serves as a captureantibody which is directed against the α-helical central fragment and asecond monoclonal antibody VIM-3B4 serves as the detector antibody whichis directed against another epitope of the α-helical central fragmentwhich is independent and different from the first epitopes. The captureantibody (GP 8) dissolved in 50 mmol/l NaHCO₃ (pH 9.6) is coated onmicrotitre plates (150×10⁻⁶ l per well) at a concentration of 10×10⁻⁶g/ml. A purified vimentin fragment at concentrations of 10 ng/ml to 500ng/ml (dissolved in buffer: 150 mmol/l NaCl, 10 mmol/l Na₂ HPO₄, pH 7.4,0.05% Tween 20) is used for the standard curve. In order to measure thevimentin concentration in the centrifuged supernatant, this is diluted1:100 to 1:500 with the latter buffer. The detector antibody VIM 3B4labelled with peroxidase is diluted to a concentration of 0.5×10⁻⁶ g/mlwith buffer (150 mmol/l NaCl, 10 mmol/l Na₂ HPO₄, pH 7.4, 1% bovineserum albumin, 0.05% Tween 20) and 150×10⁻⁶ l is used per well. Thesubstrate used is o-phenylenediamine or ABTS(2,2'-azino-di[3-ethylbenzthiazoline sulfonate (6)]). The vimentin valueobtained in this way serves as a reference value for the quantitativeevaluation of the other measurement results.

9.3 Determination of cytokeratins and detemination of cytokeratincontents.

The cytokeratins which are present in the centrifuged supernatant aredetermined immunologically by a sandwich ELISA. A first monoclonalantibody K_(s) pan 1-8, the so-called capture antibody, which isdirected against a first epitope which is typical for the cytokeratins 1to 8 is used for this. The capture antibody K_(s) pan 1-8 dissolved in50 mmol/l NaHCO₃ (pH 9.6) is coated on microtitre plates (150×10⁻⁶ l perwell) at a concentration of 2×10⁻⁶ g/ml. The purified cytokeratinfragments are for example used for the standard curve in thecombinations 8:18 and 8:20 at concentrations of 5 ng/ml to 500 ng/ml(dissolved in buffer: 150 mmol/l NaCl, 100 mmol/l Na₂ HPO₄, pH 7.4,0.05% Tween 20). K_(s) 18-27 IV and K_(s) 18-9B1 (for cytokeratin 18fragments) and IT guinea-pig antiserum (for cytokeratin 20 fragments)are used for example as peroxidase-coupled detector antibodies. In orderto measure the cytokeratin concentration in the centrifuged supernatantthis is diluted 1:100 with buffer (150 mmol/l NaCl, 10 mmol/l Na₂ HPO₄,pH 7.4, 0.05% Tween 20).

For the standardization, known amounts of the cytokeratin standardobtained according to example 7 are subjected to the sandwich ELISA. Theenzyme activity which corresponds to the concentration of each of thestandardized cytokeratins is plotted against concentration in order toobtain a standard curve from which the concentration of an unknownamount of each of the cytokeratins can be interpolated.

The extent of carcinoma metastases can be expressed by the ratio of thedetermined cytokeratin and the measured vimentin in the tissue sample.

EXAMPLE 10 Quantitative Determination of Cytokeratin 8:20 in a SandwichELISA on Microtitre Plates

10.1 Coating of microtitre plates

0.2×10⁻⁶ g capture antibody K_(s) pan 1-8 (dissolved in 100-150×10⁻⁶ l50 mmol/l sodium carbonate buffer, pH 9.6) is pipetted into each well.The plate is covered and incubated overnight at 4° C.

10.2 Washing and blocking

The excess antibody solution is removed from each well by aspiration.Three times in succession 200×10⁻⁶ l washing buffer (PBS-Tween: 150mmol/l NaCl, 10 mmol/l sodium phosphate buffer pH 7.4, 0.05% Tween 20)is pipetted into each well and removed by inverting the plate. Residualmoisture is removed by gently tapping the plate on several layers ofpaper tissues.

Each well is filled with 200×10⁻⁶ l blocking buffer (150 mmol/l NaCl, 10mmol/l sodium phosphate buffer pH 7.4, 0.05% Tween 20, 1% bovine serumalbumin, 5% sucrose; for longer storage periods 0.01% thimerosal is alsoadded) and incubated for at least 1 hour at room temperature.

10.3 Incubation with antigen or serum samples

Standard protein of cytokeratin 8:20 obtained according to example 7 atconcentrations between 5 ng/ml and 500 ng/ml (depending on therespective detector antibody) is taken up in control serum (Monitrolfrom Merz & Dade or Kontrollogen L and LU from Behring). The controlserum is used at a dilution of 1:10 and 1:100. 100×10⁻⁶ l standardprotein solution or serum samples (diluted 1:10 and 1:100) is pipettedper well and incubated for 90 min at room temperature. Afterwards it iswashed 4 times with 200×10⁻⁶ l washing buffer (PBS-Tween as describedabove)

10.4 Incubation with detector antibody

Detector antibody (CK 20 guinea-pig antiserum) coupled to peroxidase isdiluted in buffer (150 mmol/l NaCl, 10 mmol/l sodium phosphate buffer,pH 7.4, 1% bovine serum albumin) (the optimal concentration is 0.2-0.5U/ml), 100×10⁻⁶ l of this is pipetted into each well of the microtitreplate and incubated for 90 min at room temperature. Afterwards it iswashed twice with 200×10⁻⁶ l washing buffer (PBS-Tween as describedabove) and four times with 200 μl distilled water.

10.5 Substrate reaction

For a microtitre plate, 1 substrate tablet (10 mg) o-phenylenediamine(Sigma Co.) and 10×10⁻⁶ l 30% H₂ O₂ is either dissolved in 10 ml 0.1Mpotassium phosphate buffer (pH 6.0) or in citrate-phosphate buffer(0.0347 mol/l citric acid, 0.0667 mol/l disodium hydrogen phosphate; pH5.0) (when using citrate-phosphate buffer higher absorbances areobtained). 100×10⁻⁶ l substrate solution (incubated at room temperature)is pipetted into each well. The microtitre plate is covered in order toprotect the reaction from light (using aluminium foil or the like) andincubated (15-30 min) until an appropriate colour intensity hasdeveloped.

10.6 Stopping of the enzyme reaction

The peroxidase reaction is stopped by addition of 50×10⁻⁶ l 12.5% H₂ SO₄solution. In quantitative determinations the reaction for standardprotein and test serum should be stopped after the same period.

19.7 Evaluation

The microtitre plates are measured at 492 nm in an ELISA photometer.

EXAMPLE 11

10 μg standard protein (cytokeratin 20 and cytokeratin 8 reconstitutedto form filaments from a stock solution of 0.5 mg/ml in 4 mol/l urea, 10mmol/l Tris-HCl, pH 7.6, 2 mmol/l dithioerythritol, 5 mmol/l EDTA),dissolved in 100 μl phosphate-buffered saline solution (PBS; 150 mmol/lNaCl, 10 mmol/l sodium phosphate, pH 7.4) is pipetted into each well ofa 96-well microtitre plate (e.g. M 129A, Dynatech, Plochingen) andincubated for 16 h at room temperature (RT). Afterwards the wells areemptied, each is washed once with 200 μl PBS and non-saturated bindingsites are blocked by incubating for 1 hour at room temperature with 100μl 1% BSA solution (bovine serum albumin dissolved in PBS). Subsequentlythe wells are washed three times with 200 μl washing solution each time(0.05% Tween 20, dissolved in PBS), incubated for 1 hour at roomtemperature with 100 μl patient serum (diluted 1:500 in PBS), washedthree times with 200 μl washing solution each time and incubated for 1hour at room temperature with a peroxidase-coupled anti-human IgMantiserum (rabbit anti-human Ig, μ chain specific; Dako P 322) for thedetection of specific IgM antibodies and with a peroxidase-coupledanti-human IgG antiserum (rabbit anti-human Ig, gamma chain specific;Dako P 214) for the detection of specific IgG antibodies each beingdiluted 1:1000 in a 0.1% BSA solution (in PBS). After washing threetimes with 200 μl washing solution in each case and twice with 200 μldistilled water in each case, 100 μl substrate solution (10 mgo-phenylenediamine, 10 μl H₂ O₂ : 30% in 10 ml citrate-phosphate buffer:0.0347 mol/l citric acid, 0.0667 mol/l disodium hydrogen phosphate; pH5.0) is pipetted into each well. It is developed for 10-30 min(depending on the colour intensity) in darkness, the enzyme reaction isstopped by addition of 50 μl 12.5% H₂ SO₄ solution and the convertedsubstrate is measured at a wavelength of 492 nm with the aid of an ELISAphotometer. Parallel to this reference sera with a low, medium and hightitre of cytokeratin 20 autoantibodies are measured for thestandardization.

EXAMPLE 12 Immunohistochemical Localization of CK 20 (Protein IT) inNormal and Tumour Tissue of Humans

The examination methods were carried out according to known methods suchas those cited for example in example 6.

    ______________________________________                                        1. Normal tissue                                                              Epithelial cells:                                                             gastric mucous membrane (foveolae epithelium)                                                              +++                                              small intestinal mucosa      +++                                              colonic mucosa               +++                                              epithelium of the urinary tract                                                                            +++                                              Merkel cells                 +++                                              gallbladder mucosa           +                                                thymic reticulum             +                                                prostate                     +                                                liver                        -                                                pancreas                     -                                                kidney                       -                                                epidermis                    -                                                perspiratory glands          -                                                sebaceous glands             -                                                mammary gland                -                                                salivary gland               -                                                mucous membrane of the mouth -                                                oesophageal mucosa           -                                                thyroid gland                -                                                lung                         -                                                mesothelium                  -                                                uterus                       -                                                fallopian tube               -                                                epididymis                   -                                                Non-epithelial tissue: all of them negative                                   2. Tumours                                                                    Carcinomas:                                                                   adenocarcinoma of the colon  +++                                              adenocarcinoma of the stomach                                                                              ++                                               adenocarcinoma of the pancreas                                                                             ++                                               adenocarcinoma of the gallbladder                                                                          ++                                               urothelial carcinoma         +++                                              Merkel cell carcinoma        +++                                              adenocarcinoma of the lung   -*                                               mammacarcinoma               -                                                adenocarcinoma of the endometrium                                                                          -*                                               adenocarcinoma of the ovary  -**                                              adenocarcinoma of the kidney -                                                thyroid gland carcinoma      -                                                squamous cell carcinoma of the mouth cavity                                                                -*                                               squamous cell carcinoma of the lung                                                                        -*                                               squamous cell carcinoma of the cervix                                                                      -                                                small-cell carcinoma of the lung                                                                           -*                                               All non-epithelial tumours are negative                                       ______________________________________                                         + = positive                                                                  ++ = strongly positive                                                        +++ = very strongly positive                                                  - = negative                                                                  -* = negative, single cells are, however, sometimes positive                  -** = negative, except mucous ovarian carcinoma (++)                     

The immunohistochemistry was carried out with specific antibodiesagainst CK 20 (from guinea-pig or mouse) and peroxidase-coupledsecondary antibodies (anti-mouse or anti-guinea-pig Ig from the goat) orperoxidase-coupled protein A on cryostat tissue sections.

In principle this method can also be applied to paraffin sections (whenusing the mouse antibody).

The cell lines ATCC HTB 38, ATCC CCC 229, ATCC CCC 233, ATCC CCC 221 andATCC CCC 220 set forth in the description are available to anyone in thecollection of cell lines of the American Type Culture Collection,Rockville, Md., U.S.A. (ATCC) and are listed in the ATCC catalogue.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 30                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GluLysMe tPheMetGlnAsnLeuAsnAspXaaLeuAlaSerTyrLeu                             151015                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D ) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ValGlySerGluLysValThrMetGlnAsnLeuAsnAspArgLeuAla                              151015                                                                        SerTyrLeuAspLy sVal                                                           20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerGlyAsnGluLysIleThrMetGlnAsnLeuAsn AspArgLeuAla                             151015                                                                        SerTyrLeuAspLysVal                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaGlyAsnGluLysLeuThrMetGlnAsnLeuAsnAspArgLeuAla                              151015                                                                        Se rTyrLeuAspLysVal                                                           20                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ThrGlyAsnGluLysIleThrMet GlnAsnLeuAsnAspArgLeuAla                             151015                                                                        SerTyrLeuGluLysVal                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                     (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       SerGlyAsnGluLysValThrMetGlnAsnLeuAsnAspArgLeuAla                              1510 15                                                                       SerTyrLeuAspLysVal                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       IleGlnAsnGlu LysGluThrMetGlnSerLeuAsnAspArgLeuAla                             151015                                                                        SerTyrLeuAspLysVal                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17..20                                                          (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                      GluValGlnIleLysGlnTrpTyrGluThrAsnAlaProArgAlaGly                              151015                                                                        XaaXaaXaaXaaArgAspTyrSerAlaTyrTyr ArgGlnIleGlu                                202530                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                        (B) LOCATION: 14                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 18..20                                                          (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GluValLysIleArgAspTrpTyrGlnArgGlnArgProXaaAlaGlu                              151015                                                                        IleXaa XaaXaaLysAspTyrSerAlaTyrPheLysThrIleGlu                                202530                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 14                                                              (D) OTHER INFORMATION: /note="Xaa idicates that the                           amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 18..20                                                           (D) OTHER INFORMATION: /note="Xaa indicates that the                         amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GluValLysIleArgAspTrpTyrGlnArgGlnArgProXaaSerGlu                              1510 15                                                                       IleXaaXaaXaaLysAspTyrSerProTyrPheLysThrIleGlu                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                     (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 14                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                        (B) LOCATION: 18..19                                                          (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GluValLysIleHisAspTrpTyrGlnLysGlnThrProXaaAlaSer                              1 51015                                                                       ProXaaXaaGluCysAspTyrSerGlnTyrPheLysThrIleGlu                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 14                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                              between the sequences can be shown more clearly."                            (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 18..20                                                          (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GluValLysIleArgAspTrpTyrGlnLy sGlnGlyProXaaGlyPro                             151015                                                                        SerXaaXaaXaaArgAspTyrSerHisTyrTyrThrThrIleGln                                 202 530                                                                       (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 14                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                           amino acid has been ommitted so that correlations                            between the sequences can be shown more clearly."                             (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 18..19                                                          (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      Gl uValLysIleArgAspTrpHisLeuLysGlnSerProXaaAlaSer                             151015                                                                        ProXaaXaaGluArgAspTyrSerProTyrTyrLysThrIleGlu                                  202530                                                                       (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                          (B) LOCATION: 14                                                             (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GluGlyLysIleLysGluTrpTyrGluLysHisGlyAsnXaaSerHis                              15 1015                                                                       GlnGlyGluProArgAspTyrSerLysTyrTyrLysThrIleAsp                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 14..16                                                          (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                              (ix) FEATURE:                                                                (A) NAME/KEY: Peptide                                                         (B) LOCATION: 19..20                                                          (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GluSerLysIleArgGluHisHisGluLysLysGlyProXaa XaaXaa                             151015                                                                        GlnValXaaXaaArgAspTrpSerHisTyrPheLysThrIleGlu                                 2025 30                                                                       (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GluValAsnAlaAlaProGlyLeuAsnLeuGlyValIleMetAsnGlu                              1 51015                                                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ValAsnValGluMe tAspAlaAlaProGlyValAspLeuSerArgIle                             151015                                                                        LeuAsnGluMetArgAsp                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ValAsnValGluMetAspAlaAlaProGlyValAspLeuThrArgIle                              1510 15                                                                       LeuAlaGluMetArgGlu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      Va lSerValGluValAspSerAlaProGlyThrAspLeuAlaLysIle                             151015                                                                        LeuSerAspMetArgSer                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ValAsnValGluMetAspAlaThrProGlyIleAspLeuThrArgVal                              15 1015                                                                       LeuAlaGluMetArgGlu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ValAsnValGluMetAsnAlaAlaProGlyValAspLeuThrGlnLeu                              151015                                                                        LeuAsnAsnMetArgSer                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      LeuThrValGluValAspAlaProLysSerGlnAspLeuSerIleIle                              1 51015                                                                       MetAlaAspIleArgAla                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii ) MOLECULE TYPE: peptide                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GluLysGluLeuGlnSerLysLeuSerValLysAlaThrGlnLeu                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       GlnAsnLeuGluIleGluLeuGlnSerGlnLeuSerMetLysAlaSer                             151015                                                                        XaaLeuGluAsnSer                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                              between the sequences can be shown more clearly."                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GlnGlyLeuGluIleGluLeuGlnSerGlnLeuSerMetLysAlaSer                              151015                                                                        XaaLeuGluAsnSer                                                                20                                                                           (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                           amino acid has been ommitted so that correlations                            between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlnGluLeuGluIleGluLeuGlnSerGlnLeuSerMetLysAlaGly                              151015                                                                         XaaLeuGluAsnSer                                                              20                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17                                                               (D) OTHER INFORMATION: /note="Xaa indicates that the                         amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GlnGlyLeuGluIleGluLeuGlnSerGlnLeuSerMetLysAlaAla                              15 1015                                                                       XaaLeuGluAspThr                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                        (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GlnGlyLeuGluIleGluLeuGlnSerGlnLeuSerMetLysAlaGly                              1 51015                                                                       XaaLeuGluAsnThr                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii ) MOLECULE TYPE: peptide                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GlnAlaLeuGluIleGluLeuGlnSerGlnLeu AlaLeuLysGlnSer                             151015                                                                        XaaLeuGluAlaSer                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note="Xaa indicates that the                          amino acid has been ommitted so that correlations                             between the sequences can be shown more clearly."                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GlnSerLeuGl uIleArgLeuAspArgMetArgAsnLeuLysAlaSer                             151015                                                                        XaaLeuGluAsnSer                                                               20                                                                        

We claim:
 1. A process for the purification of cytokeratin 20, CK 20,comprising the steps of:producing a cytoskeletal fraction from cellscontaining CK 20, separating any proteins present by gelelectrophoresis, chromatography or by gel electrophoresis andchromatography, and isolating CK 20 from the gel or from achromatographic fraction containing the CK
 20. 2. The process accordingto claim 1, wherein said cytoskeletal fraction is produced from duodenalmucosal villi.
 3. The process according to claim 1, wherein saidcytoskeletal fraction is produced from culture cells which are derivedfrom carcinomas selected from the group consisting of colon carcinomas,bladder carcinomas and gastric carcinomas.
 4. The process according toclaim 1, wherein said proteins are separated bycarrying out a first SDSpolyacrylamide gel electrophoresis in a buffer system with an increasedsalt concentration, cutting out any band containing CK 20, eluting theCK 20 from said band, subjecting said CK 20 to a second SDSpolyacrylamide gel electrophoresis in a buffer system with low saltconcentration, and isolating purified CK 20 from any corresponding bandin the gel.
 5. The process according to claim 1, wherein said proteinsare separated by chromatographic fractionation comprising the stepsof:carrying out anion exchange chromatography; and subsequentlysubjecting any fractions containing CK 20 to HPLC.
 6. The processaccording to claim 5, wherein said anion exchange chromatography iscarried out on DEAE-cellulose in the presence of urea using an eluantwith a linear gradient of between 0 and 100 mmol/l guanidiniumhydrochloride.
 7. The process according to claim 6, wherein thefractions containing CK 20 contain 38 to 50 mmol/l guanidiniumhydrochloride.
 8. A protein material comprising a reconstitutedcytokeratin complex containing CK 20 and a basic cytokeratin selectedfrom the group consisting of cytokeratins 1 to 8 or correspondingα-helical central fragments of CK 20 and said basic cytokeratin preparedby proteolytic cleavage.
 9. The protein material according to claim 8,wherein said basic cytokeratin is CK 8 or α-helical central fragments ofCK
 8. 10. A process for the production of a standard protein materialwhich contains CK 20 and a basic cytokeratin, comprising the stepsofdissolving purified CK 20 and a purified basic cytokeratin selectedfrom the group consisting of cytokeratins 1 to 8 in an equimolar ratioin a buffer containing urea to produce a mixture, dialyzing the mixtureagainst a first buffer containing urea and DTT, and then dialyzing themixture against a second buffer without urea.
 11. The process accordingto claim 10, wherein any cytokeratin complex which forms is subsequentlycleaved proteolytically.
 12. The process according to claim 11, whereinsaid proteolytic cleavage is carried out with chymotrypsin in an enzymeto substrate ratio of 6:1000 to 10:1000 with a digestion period ofbetween 30 and 60 minutes.
 13. The process according to claim 10,wherein said purified basic cytokeratin is CK
 8. 14. The processaccording to claim 10 wherein said CK 20 is purified according toclaim
 1. 15. The process according to claim 10, wherein said purified CK20 and purified basic cytokeratin are dissolved in a buffer containing8.5 to 10 mol/l urea and 1.5 to 3 mmol/l DTT, and said first buffercontains 3.5 to 4.5 mmol/l urea and 1.5 to 3 mmol/l DTT.
 16. A processfor the production of antibodies specific for CK 20, comprising thesteps of:producing a cytoskeletal fraction from cells containing CK 20,separating any proteins present by gel electrophoresis, chromatographyor by gel electrophoresis and chromatography, isolating CK 20 from thegel or from the chromatographic fraction containing the CK 20, thenimmunizing a suitable animal with said CK 20, and isolating anypolyclonal or monoclonal antibodies produced.
 17. The process accordingto claim 16, wherein after the production of polyclonal antibodies,monospecific antibodies are isolated by immunoprecipitation andseparation of antibodies directed against other cytokeratins orimmunoprecipitation and isolation of the antibodies specific for CK 20.18. The process according to claim 17, wherein said polyclonal ormonoclonal antibodies are incubated with a solid phase to which CK 20has been coupled.
 19. The process according to claim 17, wherein saidpolyclonal or monoclonal antibodies are incubated with a solid phase towhich electrophoretically purified cytokeratins 8, 18 and 19 or proteinfrom cells containing cytokeratins 8, 18 and 19 have been coupled. 20.The process according to claim 17, wherein several immunoprecipitationsteps are carried out.
 21. The process according to claim 18, whereinsaid solid phase is nitrocellulose strips.
 22. The process according toclaim 19, wherein said solid phase is nitrocellulose strips.
 23. Aprocess for the immunological identification of CK 20 or its α-helicalcentral fragment obtained by proteolytic cleavage, in a sample selectedfrom the group consisting of tissue sections, tissue homogenates andbody fluids, comprisingcontacting said sample with an antibody specificfor CK 20 or its α-helical central fragment, and determining anyantibody-antigen complexes formed, wherein said antibody is preparedaccording to claim
 16. 24. The process according to claim 23, furthercomprisingforming a homogenate of said sample, proteolytically cleavingany intermediary filament proteins present in said homogenate to releaseany α-helical central fragments present into the soluble phase,isolating said α-helical central fragments, and identifying andquantitatively determining said fragments using said antibody.
 25. Theprocess according to claim 23, wherein said sample contains solubleintermediary filament protein fragments present in body fluids selectedfrom the group consisting of blood, blood serum and urine.
 26. A methodfor the detection of autoantibodies against CK 20 in blood or serumcomprising adding a protein material comprising a reconstitutedcytokeratin complex containing CK 20 and a basic cytokeratin selectedfrom the group consisting of cytokeratins 1 to 8 or correspondingα-helical central fragments prepared by proteolytic cleavage of CK 20and said basic cytokeratin, to a blood or serum sample and detecting anyautoantibodies which bind to said cytokeratin complex.
 27. A method fordistinguishing carcinomas of the gastrointestinal tract, the bladder andMerkel cells from other tumors, or for testing for the cellular originof metastases, comprising determining the presence of CK 20 in a tissueto be examined with antibodies specific for CK
 20. 28. The methodaccording to claim 27, wherein said tumors are other carcinomas.
 29. Aprocess for the production of antibodies specific for CK 20, comprisingthe steps of:producing a cytoskeletal fraction from cells containing CK20, separating any proteins present, isolating CK 20, then immunizing asuitable animal with said CK 20, and isolating any polyclonal ormonoclonal antibodies produced.